YAC and Cosmid Contigs Encompassing the Fukuyama-Type Congenital Muscular Dystrophy (FCMD) Candidate Region on 9q31

GENOMICS

40, 284–293 (1997) GE964584

ARTICLE NO.

YAC and Cosmid Contigs Encompassing the Fukuyama-Type Congenital Muscular Dystrophy (FCMD) Candidate Region on 9q31 MASASHI MIYAKE, YUTAKA NAKAHORI, IKUMI MATSUSHITA, KAZUHIRO KOBAYASHI, KUNIHIKO MIZUNO, MOMOKI HIRAI,* ICHIRO KANAZAWA,† YASUO NAKAGOME, KATSUSHI TOKUNAGA, AND TATSUSHI TODA1 Department of Human Genetics, Graduate School of International Health, *Department of Biological Sciences, Graduate School of Science, and †Department of Neurology, Faculty of Medicine, University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113, Japan Received June 24, 1996; accepted December 16, 1996

Fukuyama-type congenital muscular dystrophy (FCMD), the second most common form of childhood muscular dystrophy in Japan, is an autosomal recessive severe muscular dystrophy associated with an anomaly of the brain. We had mapped the FCMD gene to an approximately 5-cM interval between D9S127 and D9S2111 on 9q31–q33 and had also found evidence for linkage disequilibrium between FCMD and D9S306 in this candidate region. Through further analysis, we have defined another marker, D9S172, which showed stronger linkage disequilibrium than D9S306. A yeast artificial chromosome (YAC) contig spanning 3.5 Mb, which includes this D9S306–D9S172 interval on 9q31, has been constructed by a combination of sequence-tagged site, Alu-PCR, and restriction mapping. Also, cosmid clones subcloned from the YAC were assembled into three contigs, one of which contains D9S2107, which showed the strongest linkage disequilibrium with FCMD. These contigs also allowed us to order the markers as follows: cen–D9S127–(Ç800 kb)–D9S306 (identical to D9S53)–(Ç700 kb)–A107XF9– (Ç500 kb)–D9S172–(Ç30 kb)–D9S299 (identical to D9S774)–(Ç120 kb)–WI2269–tel. Thus, we have constructed the first high-resolution physical map of the FCMD candidate region. The YAC and cosmid contigs established here will be a crucial resource for identification of the FCMD gene and other genes in this region. q 1997 Academic Press

INTRODUCTION

Fukuyama-type congenital muscular dystrophy (FCMD; MIM No. 253800), the second most common form 1

To whom correspondence should be addressed at Department of Human Genetics, Graduate School of International Health, University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113, Japan. Telephone: /81-33812-2111. Fax: /81-3-5802-2907. E-mail: [email protected] until April 1997. After April 1997 correspondence should be addressed to this author at Laboratory of Genome Medicine, Institute of Medical Science, University of Tokyo, 4-6-1 Shiroganedai, Minato-ku, Tokyo 108, Japan. Telephone: /81-3-5449-5376. Fax: /81-3-5449-5433. E-mail: toda@ ims.u-tokyo.ac.jp.

0888-7543/97 $25.00 Copyright q 1997 by Academic Press All rights of reproduction in any form reserved.

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of childhood muscular dystrophy in Japan, is an autosomal recessive severe congenital muscular dystrophy associated with an anomaly of the brain (Fukuyama et al., 1981). The incidence of FCMD is 7–12/100,000, and 1 in 100 persons is presumed to be a heterozygous carrier (Fukuyama and Ohsawa, 1984). Patients with FCMD manifest weakness of facial and limb muscles and general hypotonia, which appears before 9 months of age. Functional disabilities are more serious in patients with FCMD than in Duchenne muscular dystrophy patients; usually the maximum motor function is shuffling, and most patients are never able to walk without support. Simultaneously they exhibit severe mental and speech retardation, and they need careful nursing as there is no effective therapy. Patients usually become bedridden before 10 years of age because of generalized muscle atrophies and joint contracture, and most of them die by the age of 20 (Fukuyama et al., 1981). Analysis of Duchenne muscular dystrophy has contributed to the understanding of muscular dystrophy in general. Its protein product, dystrophin, is a large rodlike component of the membrane cytoskeleton of muscle fiber and is known to be associated with a large oligomeric complex of sarcolemmal glycoproteins (dystrophin-associated proteins; DAPs) (Ervasti and Campbell, 1991). Recent studies have revealed that defects in DAPs are responsible for various muscular dystrophies (Worton, 1995). It was once reported that expression of one of the DAPs, b-dystroglycan, is abnormally low in FCMD muscle, based on immunohistochemical analysis (Matsumura et al., 1993), as well as a significant reduction in immunostainning of an extracellular matrix, laminin a2 (merosin) (Hayashi et al., 1993). Genes encoding these proteins are located on chromosomes 3p21 (Igbraghimov-Beskrovnaya et al., 1993) and 6q22–q23 (Tryggvason, 1993), respectively. However, we localized the FCMD locus around D9S58 on chromosome 9q31–q33 using genetic linkage analysis and homozygosity mapping (Toda et al., 1993).

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Subsequently, the gene candidate region was narrowed to approximately 5 cM between loci D9S127 and D9S2111 (CA246) by homozygosity and recombination mapping, and evidence was found for linkage disequilibrium between FCMD and the 9q31 locus, D9S306 (mfd220), in this candidate region. We presumed that the FCMD gene could lie within 1 Mb of D9S306 based on precedents such as cystic fibrosis and Huntington disease (Toda et al., 1994, 1995) (Fig. 1). Through further analysis, we found another marker, D9S172, that is in linkage disequilibrium with FCMD. To facilitate the identification of the FCMD gene, we constructed 3.5 Mb of a yeast artificial chromosome (YAC) contig that includes the D9S306–D9S172 interval on 9q31 and its physical map. Cosmid clones subcloned from YAC were assembled into three contigs. One of these included the marker D9S2107 (Toda et al., 1996), which is presumed to be the closest to the FCMD gene by linkage disequilibrium mapping. Both YAC and cosmid contigs provide crucial resources for the isolation of the FCMD gene and unknown genes in this candidate region. MATERIALS AND METHODS DNA typing. Sixty-three FCMD families and 60 unrelated Japanese individuals were genotyped with polymorphic microsatellites D9S306 (Weber, 1993), A107XF9 (Weissenbach, 1995), D9S172 (Gyapay et al., 1994), and D9S299 (Cooperative Human Linkage Center et al., 1994). PCR was performed as described previously (Toda et al., 1994). Screening of YAC clones. YAC DNA pools from the CEPH YAC library (Bellanne´-Chantelot et al., 1992) were screened by PCR with D9S306 according to the three-dimensional procedure, and the YAC clones that contain D9S172 were identified through the CEPH YAC database [QUICKMAP (Cohen et al., 1993)]. The chromosomal localization and chimerism of YAC clones used for restriction mapping were verified by fluorescence in situ hybridization (FISH) to metaphase chromosomes. YAC contig based on sequence-tagged sites (STSs) and Alu-PCR. Isolated YAC clones were examined for the presence or absence of the following markers: D9S277 and D9S271 (Gyapay et al., 1994); D9S109 (Furlong et al., 1992); D9S127 (Lyall et al., 1992); D9S306; A107XF9; D9S172; D9S299; WI2269 (Hudson et al., 1995); and D9S748 (Povey et al., 1994), by PCR with each recommended condition. Alu-PCR was carried out using four primers: 938 (Hirst et al., 1991), 939 (5*-GTGCTGGGATTACAGGCGTG-3*), Alu-S (5*-GAGGTTGCAGTGAGCCGAGAT-3*), and Alu-J (5*-GAGGCTGCAGTGAGCCGTGAT-3*), separately. Reaction conditions were 3 min at 957C, 2 min at 507C, and 2 min at 727C followed by 36 cycles of 1 min at 957C, 2 min at 507C, and 2 min at 727C. Pulsed-field gel electrophoresis (PFGE) analysis. YAC DNA was prepared in agarose blocks as described previously (Imai et al., 1990). The positions of the restriction sites for NotI, MluI, and NruI were determined by complete single and double digestions, and that for BssHII was determined by partial digestion. Digested samples were separated through 1% agarose gels in 0.251 TBE buffer using a LKB Pulsaphor unit (Pharmacia). Electrophoresis was carried out using two run conditions; fragments of 50– 600 kb were resolved by an interval of 20 s for 20 h, 190 V, 47C, and fragments of 150 kb to over 1 Mb were resolved by an interval of 90 s for 40 h, 150 V, 47C. Gels were blotted onto Hybond-N/ nylon membrane (Amersham). Filters were hybridized with total human DNA to detect all frag-

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ments and with the PCR amplified left arm (L1: 5*-CTCATGTTTGACAGCTTATC-3*; L2: 5*-TGATCGCGTAGTCGATAGTGC-3*) and right arm (R1: 5*-TCTACGCCGGACGCATC-3*; R2: 5*-GTCATAAGTGCGGCGACG-3*) of the pYAC4 vector as probes to detect end fragments. YAC end isolation. A ‘‘vectorette PCR method’’ was used to isolate YAC insert ends. Primer sequences and experimental conditions were as described by Reley et al. (1990). End products were digested with EcoRI to remove the vector region and separated through a 2% agarose gel. Construction of a cosmid library from y913B5. YAC high-molecular-weight DNA was prepared as described previously (Davis et al., 1986). Cell lysate was fractionated on a sucrose step gradient and examined by PFGE. YAC DNA (1–2 mg) was partially digested with Sau3AI to subclone into the BamHI site of the SuperCos1 vector (Stratagene). Ligation of inserts to SuperCos1 and packaging with Lambda Inn (Nippon gene) were performed as recommended by the manufacturers. Subclones that have inserts from human genomic DNA were selected by colony hybridization with total human DNA / CotI DNA (GibcoBRL) labeled radioactively. A total of 164 positive clones (fivegenome-equivalent) were obtained. Cosmid DNA was isolated by the alkaline lysis method (Sambrook et al., 1989). Regional assignment of the cosmids containing either each marker or rare-cutting restriction sites on YACs. Cosmids containing markers D9S306, A107XF9, D9S172, and D9S299 were isolated by colony hybridization with one end-labeled primer of each microsatellite marker as probes from the YAC-derived cosmid library. Positive clones were confirmed for the presence of each marker by PCR. Also, cosmid clones containing either NotI or NruI were selected by digestion with these restriction enzymes. These cosmids, which contain each marker or restriction sites, were digested with NotI to separate insert DNAs. A labeled insert DNA was hybridized to the filters that were used for the YAC restriction mapping. Construction of cosmid contigs. Cosmid contigs were constructed mainly using the method described by Murata et al. (1994). A total of 164 clones, equivalent to nearly five genomes, were digested with EcoRI. First, 100–200 ng of each DNA was electrophoresed in a 0.8% agarose gel to confirm complete digestion. A small amount (Ç10 ng) of each sample was reelectrophoresed and transferred onto HybondN/ nylon filters. Membranes were probed with individual whole cosmids (10 ng) by competitive hybridization to reveal shared fragments. Hybridizations at a relatively low radioactivity (1 1 105 cpm/ml hybridization solution) were performed to reduce nonspecific signals by Alu–Alu annealing. Cosmids that shared two or more bands with a probe cosmid were considered to be contiguous. By repeating this experiment, contigs were expanded to both sides of anchor cosmids. Also, T3 or T7 walking was performed using cosmid end RNA probes (RNA transcription kit; Stratagene). Hybridizations. Membranes were prehybridized in 10% SDS and 7% PEG containing 200 mg/ml sonicated human placental DNA at 657C overnight. Probe DNA was radiolabeled with [a-32P]dATP using the Megaprime labeling kit (Amersham), prehybridized in the same solution at 657C for 1 h, and hybridized with the membranes (Tokino et al., 1991). The final washing stringency was 0.11 SSC, 0.1% SDS at 657C. For an oligonucleotide probe end-labeled with [g-32P]ATP, the hybridization and final wash were performed in 61 SSC, 11 Denhardt’s, 0.1% SDS at (Tm 0107C) and in 61 SSC, 0.1% SDS at (Tm 0 57C), respectively.

RESULTS

Construction of a YAC Contig Spanning the FCMD Locus We previously placed FCMD distal to D9S127 by recombination mapping and found the marker, D9S306 (mfd220), that showed linkage disequilibrium with

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Then we selected three clones, y930D3, y747D8, and y913B5, that were most efficient for the construction of a YAC contig covering the FCMD candidate region. These YAC clones were used for the following analyses of restriction mapping and construction of the cosmid contig. Long-Range Restriction Mapping

FIG. 1. Physical and genetic locations of markers on chromosome 9q31–q33.

FCMD (Toda et al., 1994) (Fig. 1). We further found that another marker, D9S172, is also in linkage disequilibrium (Table 1). Since linkage disequilibrium with a disease is observed in very close markers, both loci, D9S306 and D9S172, were used as anchor points to construct a YAC contig spanning the region that is predicted to contain the FCMD gene. A total of 10 YAC clones were isolated from the CEPH mega-YAC library. YAC clones y930D3, y887B2, y974B2, y759F2, y747D8, and y786A3 contained D9S306. Using QUICKMAP, other YAC clones, y886H1, y959D1, y913B5, and y802A1, were found to contain D9S172 or were localized to the D9S306–D9S172 interval (Table 2). We constructed a rough contig using a combination of STS content and Alu-PCR mapping. First, the 10 YAC clones were examined for the presence or absence of the markers in the map. Table 2 shows the results of STS content mapping. y930D3 contained two loci, D9S127 and D9S306. Also, y959D1, y913B5, and y802A1 included four loci, A107XF9, D9S172, D9S299, and WI2269. On the other hand, y887B2, y974B2, and y886H1 contained only one locus. There were no YAC clones encompassing two anchored loci, D9S306 and D9S172. Concurrently, Alu-PCR using the primers 938, 939, Alu-J, and Alu-S allowed us to analyze overlapping of each clone by classifying the common bands from the electrophoretic patterns of these products (Fig. 2). In this way, we constructed a rough contig that contains the D9S306–D9S172 interval: y930D3–y887B2– (y759F2, y747D8, y974B2)–y886H1–(y913B5, y802A1). Simultaneously, all YAC clones were tested for chimerism by FISH, and three YAC clones, y887B2, y786A3, and y959D1, were obviously chimeric (Table 2).

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Construction of restriction maps was performed on y930D3, y747D8, and y913B5. The positions of the restriction sites for NotI, MluI, and NruI were determined by single and double digestion combinations. For BssHII, the position was determined by partial digestion. In addition to the rough contig constructed using STS and Alu-PCR mapping, analysis of the positions of common restriction sites among overlapping clones enabled us to determine the degree of overlap more precisely (Fig. 3). Also, we generated YAC end probes using vectorette PCR to confirm that both end regions were consistent with the results of restriction mapping. However, the restriction map of the right end region of y747D8 was not consistent with that of the equivalent region of y913B5. By using the right end probe of y747D8, we could identify that this region corresponded to a 340kb MluI–MluI fragment of y913B5. We tested other end probes of three YAC clones in the same way, and the positions of overlap were reasonably consistent with restriction mapping. Thus, we were able to construct a precise YAC contig that is predicted to contain the FCMD gene (Fig. 3). Localization of the Cosmid Clones Containing Restriction Sites or Markers Linkage disequilibrium mapping has indicated the distance between each microsatellite marker and the FCMD gene locus (Toda et al., 1996). Therefore, to identify the FCMD gene, it is very important to localize each marker on the YAC contig. Then we screened cosmid clones that contain each marker from the YACderived cosmid library and isolated cosmid clones, c127 TABLE 1 Linkage Disequilibrium of FCMD with D9S172 No. of chromosomes observed PCR product size (bp)

Normal

FCMD

303 301 299 297 295 293 291

7 60 3 70 58 24 0

0 90 0 15 7 0 2

Totals

222

114

Note. x 2 Å 92.4; 6 df; P õ 0.000001.

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TABLE 2 STS Mapping of YAC Clones STS YAC

Chimerisma

Sizeb (Mb)

930D3 887B2 974B2 759F2 747D8 786A3 886H1 959D1 913B5 802A1

N Y N N N Y N Y N N

1.46 1.30 0.65 1.35 2.00 1.55 0.93 1.71 1.88 1.75

D9S271

D9S109

D9S277

D9S127

D9S306

A107XF9

D9S172

D9S299

WI2269

D9S748

0 0 0 0 0 0 0 0 0 0

0 0 0 0 0 0 0 0 0 0

0 0 0 0 0 0 0 0 0 0

/ 0 0 0 0 0 0 0 0 0

/ / / / / / 0 0 0 0

0 0 0 / / / / / / /

0 0 0 0 0 0 0 / / /

0 0 0 0 0 0 0 / / /

0 0 0 0 0 0 0 / / /

0 0 0 0 0 0 0 0 0 0

Note. Presence or absence of the respective markers tested by PCR assay is indicated by / and 0 signs. a Y, chimeric clone; N, not chimeric. b The sizes of YACs y930D3, y747D8, and y913B5 were determined by PFGE analysis. Other sizes were according to the CEPH YAC database.

(D9S127), c220 (D9S306), c747-1 (A107XF9), cJ8 (D9S172), c802-1 (D9S299), and cF1 (WI2269). Also, cosmid clones containing restriction sites cM1, cJ12, and cO6 for NotI and cF1 for NruI were identified. To assign these cosmid clones on the YAC contig,

purified insert DNAs were hybridized to the filters of PFGE-separated fragments with NotI, MluI, and NruI in single and double complete digestion. Figure 4 shows examples of regional assignment of cosmid clones cJ12 and cJ8, which contain the NotI site and D9S172, re-

FIG. 2. (A) A rough YAC contig spanning the FCMD region based on a combination of STS content and Alu-PCR mapping. (Top) Positions of the markers based on STS mapping are represented in a centromeric to telomeric order. The YACs are represented by thick solid horizontal arrows. The partitions that are divided by vertical thin lines show the common bands from the electrophoretic patterns of Alu-PCR products. (B) Electrophoretic patterns of Alu-PCR products using 939 primer. M, PHY marker.

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FIG. 3. Long-range restriction map of three YACs that are most efficient for covering the FCMD candidate region on 9q31. The remaining seven YACs, which are depicted by thick solid horizontal arrows, were positioned only by the means of STS content and Alu-PCR mapping. Detailed positions of the markers are also represented in a centromeric to telomeric order. Restriction enzyme sites shown as vertical lines are N, NotI; M, MluI; Nr, NruI; and B, BssHII. R and L correspond to pYAC4 right end and left end, respectively.

spectively. These analyses provided each marker locus on the restriction map. D9S127 maps to a 350-kb NruI– MluI fragment in y930D3. D9S306 maps to a 500-kb left end NruI fragment in y930D3 and a 600-kb NruI– NruI fragment in y747D8. A107XF9 maps to a 260-kb MluI–NotI fragment in y913B5. D9S172 and D9S299 map to a 300-kb NotI–NruI fragment in y913B5, and further, these marker loci were determined to be proximal to Ç80 kb of the NruI site of y913B5 based on the cosmid contig described below. Although WI2269 maps to a 270-kb NruI–MluI fragment in y913B5, it was also contained in a cosmid clone, cF1, which was localized to the NruI site of y913B5. Therefore, it lies very close to the NruI site of y913B5 (Figs. 3 and 6A). Although the restriction map based on genomic DNA does not always match that of YAC because of the difference in methylation between human and yeast, hybridization results of these marker cosmids to PFGE blots (NotI and BssHII) of DNA from the normal human lymphoblastoid cell line agreed with the restriction map of the YAC contig (data not shown). Assembly of the Cosmid Contigs of y913B5 Although we placed FCMD distal to D9S127 by recombination mapping (Toda et al., 1994), we further found that two families were recombinant at D9S306 (mfd220). This result placed the FCMD gene distal to D9S306 (Fig. 5). There were no recombinations between FCMD and other polymorphic markers used in this study.

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Furthermore, although we presumed that the FCMD gene could lie within 1 Mb of D9S306 based on the precedents (Toda et al., 1994), linkage disequilibrium mapping using the formula Pexcess Å (1 0 mgq01)(1 0 u)g (Lehesjoki et al., 1993) indicated that the estimated distances between FCMD and D9S306/D9S172 were approximately 1 Mb and 280 kb, respectively (Toda et al., 1996). Taking these genetic analyses and the YAC physical data together, it was suggested that the FCMD gene lies on y913B5 (Fig. 3). For the purpose of analyzing the FCMD candidate region in detail, we intended to construct cosmid contigs, using the localized cosmids that contain each marker and the NotI or NruI restriction site as anchor points to extend the contigs. First, Southern blots of EcoRI-digested cosmids were prepared, and the anchor cosmids were hybridized to these filters. We could identify the overlapping cosmids sharing one or several bands with the anchor cosmids. Cosmids demonstrating irregular hybridization patterns, a constant band with all probes, were excluded. By repeating this experiment, the contigs were effectively extended to both sides of the anchor cosmids. Then, we constructed three groups of cosmid contigs of y913B5. Contig groups I, II, and III harbored A107XF9, two NotI sites with an interval of 160 kb, and D9S172– WI2269, respectively (Figs. 6A and 6B). When the cosmid cJ8, containing D9S172, was used as a probe, 10 cosmids were identified, including previously identified cosmid clone c802-1, containing

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FIG. 4. Regional assignment of the cosmid clones cJ12 and cJ8 (D9S172) on the y913B5 restriction map. The Southern blots were hybridized with purified insert DNA of each cosmid. cJ12 contains the NotI site in the insert DNA, so two fragments were generated by NotI digestion. cJ12-S and cJ12-L show the shorter and longer NotI fragment of cJ12 insert DNA, respectively. ND, nondigested y913B5 DNA. cJ12 and cJ8 map to the telomeric NotI site and a 300-kb NotI–NruI fragment in y913B5, respectively.

FIG. 5. Recombinations at D9S306. Genotypes in two families demonstrating crossovers at D9S306 are indicated by the size of PCR products for three polymorphic microsatellite loci. Haplotypes were constructed assuming the most parsimonious linkage phase. The haplotype carrying the FCMD allele is boxed. An asterisk under family OKA96 indicates uncertainty with respect to the precise position of crossover, because of the uninformativeness of A107XF9 in the mother of this family. The observed recombination events in these families place the FCMD gene distal to D9S306.

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FIG. 6. (A) The locations of three groups (I, II, and III) of cosmid contigs on the y913B5 restriction map. The thin horizontal lines denote cosmid clones. Restriction enzyme sites shown as vertical lines are N, NotI; M, MluI; Nr, NruI; and B, BssHII. R and L correspond to pYAC4 right end and left end, respectively. (B) EcoRI-digested electrophoretic patterns of cosmid clones constituting each contig. M, 1kb ladder marker.

D9S299. Subsequently, we noticed that c802-1 contained both the D9S172 and the D9S299 loci, and the distance was estimated to be Ç30 kb, although both loci mapped to the same 300-kb NotI–NruI fragment in y913B5 (Figs. 3 and 6A). High-Resolution Restriction Map of the Group II Cosmid Contig We further noticed that the Group II cosmid contig contains the marker D9S2107 (Toda et al., 1996), which is presumed to be the closest to the FCMD gene, with an estimated distance of 20 kb by linkage disequilibrium mapping. This contig was constructed by extending from both anchored cosmids, cM1 and cJ12, and spans a 280-kb region including two NotI sites. (Figs. 6A and 7). An EcoRI restriction map of this contig was obtained by ordering the fragments based on differences in overlapping clones and by hybridization with each cosmid

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end RNA probe. To determine the locations of rarecutting restriction enzyme sites, NotI, MluI, and BssHII, cosmids were double digested with EcoRI and one of each enzyme. As a result, we noticed that these enzyme sites were concentrated around both NotI sites, which shows that this contig probably contains two potential CpG islands. We were able to define the precise location of D9S2107 based on this high-resolution EcoRI restriction map. It was contained in an 8-kb EcoRI fragment of cosmid cJ12 and also lies within a 30-kb region from the telomeric NotI site (Fig. 7). Ordering the Markers and Intermarker Distance Localization of cosmids containing each marker and construction of cosmid contigs provided the precise ordering of markers and estimates of the physical distance between loci. Interestingly, we noticed that the markers D9S306 and D9S299 are identical to D9S53 (Attwood et

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FIG. 7. Group II cosmid contig and high-resolution restriction map of a Ç280-kb region including the marker D9S2107, closest to the FCMD gene. The cosmids are represented by horizontal lines and EcoRI sites as short vertical lines. The lines stretching below are the EcoRI sites whose order have not been determined. The rare-cutting restriction enzyme sites shown as tall vertical lines are N, NotI; M, MluI; and B, BssHII.

al., 1994) and D9S774 (Povey et al., 1994), respectively, comparing each flanking sequence. Also, we found that the markers D9S271, D9S109, D9S277, and D9S748 were absent from our physical map. The order and physical distance of the genetic markers based on our physical map are cen–(D9S271, D9S109, D9S277)–D9S127– (Ç800 kb)–D9S306 (Å D9S53)–(Ç700 kb)–A107XF9– (Ç500 kb)–D9S172–(Ç30 kb)–D9S299 (Å D9S774)– (Ç120 kb)–WI2269–D9S748–tel. DISCUSSION

We have established a 3.5-Mb YAC contig of the FCMD candidate region including the D9S306– D9S172 interval on 9q31. Orientation of the contig has been confirmed by the analysis of STSs in combination with Alu-PCR analysis. Detailed restriction mapping of three YAC clones, y930D3, y747D8, and y913B5, has enabled us to determine the precise extent of overlap between the YACs. Also, cosmid clones containing each marker were localized on the restriction map. The combined results of restriction mapping and cosmid localization provided a reliable estimation of physical distance between the markers. When this work was initiated, the order of markers based on the Genome Data Base (GDB) was cen– D9S127–D9S53–D9S271–D9S109–D9S277–D9S299– D9S172–D9S748–tel. However, our STS content map and localization of each marker on YAC restriction maps provided the precise ordering of the markers from the FCMD candidate region as follows: cen–(D9S271, D9S1 0 9, D 9 S 2 7 7) – D 9 S 1 2 7 – D 9 S 3 0 6 (Å D 9 S 5 3)– A107XF9 – D9S172 – D9S299 (Å D9S774) – WI2269– D9S748–tel. We also noticed that the markers D9S306 and D9S299 are identical to D9S53 and D9S774, respectively.

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The physical distance between two markers, D9S306 and D9S172, which shows linkage disequilibrium with FCMD, is approximately 1.2 Mb. Based on linkage disequilibrium studies, we predicted that the FCMD gene should be located within 1 Mb and 280 kb of D9S306 and D9S172, respectively (Toda et al., 1994, 1996). Combined with recombination events at D9S306, we were able to estimate the rough position of the FCMD locus on the YAC contig. As a result, we predicted that y913B5 is enough to cover the FCMD gene region, and further analyses were carried out on y913B5. We have also constructed three groups of cosmid contigs derived from a y913B5-specific library. These cosmid contigs were efficiently constructed using the cosmids containing each marker and a NotI or NruI site as anchor points. Also, we developed several new microsatellite markers from cosmid clones of these contigs. We found the marker D9S2107 from a cosmid clone, cJ12, which showed the strongest linkage disequilibrium with FCMD and predicted that FCMD lies within 20 kb of D9S2107 (Toda et al., 1996). Linkage disequilibrium mapping is a powerful tool for high-resolution genetic mapping of genes in isolated founder populations such as the Finns as well as the Japanese (Jorde, 1995). Diastrophic dysplasia is one of the good illustrations that was isolated by fine-structure linkage disequilibrium mapping (Ha¨stbacka et al., 1994). In light of the example of diastrophic dysplasia, we presume that the FCMD gene exists in the Group II cosmid contig and that it lies extremely close to D9S2107. A recent review showed that disequilibrium and physical distance did not always correlate significantly when a region õ60 kb was studied but nearly always correlated significantly in larger genomic regions (Jorde et al., 1994). Furthermore, this approach is more

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useful and accurate in isolated founder populations such as the Japanese. We consider our presumption that the FCMD gene lies in the Group II cosmid contig to be reasonable. A high-resolution restriction map of the Group II cosmid contig allowed us to map the precise location of D9S2107 in this contig. It lies within a 30- and a 10kb region of the telomeric NotI site and the BssHII site, respectively. Approximately 90% of the NotI and 75% of the BssHII site correspond to a CpG island (Lindsay and Bird, 1987). Since CpG islands are indicators of expressed genes, the strong candidate region of the FCMD gene, around a 20-kb region of D9S2107, possibly contains some expressed genes. Also, there are only two NotI sites in this 3.5-Mb YAC contig. Strikingly, the other NotI site is located proximal to 160 kb of the telomeric NotI site. Other rare-cutting enzyme sites, MluI and BssHII, were also included in the NotI–NotI 160-kb region. Therefore, the region that was covered by the Group II cosmid contig seems to be relatively gene-rich in comparison with other regions of the YAC contig. Hence, screening of the transcripts from the Group II cosmid contig, using exon trapping (Buckler et al., 1991) or cDNA selection (Morgan et al., 1992), will result in the isolation of not only the FCMD gene but also other novel genes. We have presented the first physical maps of the FCMD candidate region. These physical maps should facilitate the isolation of the FCMD gene and unknown genes in this candidate region. ACKNOWLEDGMENTS We gratefully acknowledge Drs. Takako Takano, Kumiko Takadaya, and Yusuke Nakamura for their help and Ms. Ayuchi Tsuboi for reading the manuscript. This work was supported by Research Grants for Nervous and Mental Disorders (5A-2 and 7A-5) and for Pediatric Research (7C-1) from the Ministry of Health and Welfare and also by Grants-in-Aid for Scientific Research on Priority Areas (07264208) and for Scientific Research (07670699) from the Ministry of Education, Science, and Culture, Japan.

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